As an important inorganic nutrient of plants, nitrogen participates in various physiological metabolic processes, being a main component of proteins, nucleic acids and secondary metabolites. The nitrogen concentration in plants is influenced by the environmental nitrogen level. Generally, low nitrogen will reduce the concentrations of amino acids and other nitrogen-containing compounds (e.g., chlorophyll) in plants. These changes will affect the carbon and nitrogen assimilation of plants and limit the accumulation of plant biomass (Schlüter et al., 2012). Plants grown with limited resources may prefer to produce more defensive compounds (Wilkens et al., 1996). High nitrogen significantly promotes the growth of plants with adequate environmental resources, but plants in high-nitrogen environments have rich cytoplasm and thinner cuticles (Jauset et al., 2000). High nitrogen fertilization leads to a reduction in the content of lignin, which decreases plant defenses by mediating the thickness of the plant’s physical barrier (Zhang et al., 2017). Nitrogen fertilizers may not directly affect the physiology of insects. High nitrogen would shift plant physiology (e.g., amino acid biosynthesis and tricarboxylic acid cycle) to affect the nutritional conditions for herbivorous insects (Foyer et al., 2011). Herbivores prefer high-nitrogen plants, and the population size of herbivores increases significantly in high-nitrogen plants (Li et al., 2016). The population of Bemisia tabaci increases with increasing amounts of nitrogen fertilizer under field conditions (Bi et al., 2001). High nitrogen also increases the egg survival rate and pupal exuviae size of Trialeurodes vaporariorum on tomato plants (Jauset et al., 2000).

A high-nitrogen environment supplies plants with more of this macronutrient, and insects can obtain more suitable nutrients under this nutrient condition. The carbon-nutrient balance hypothesis (Bryant et al., 1983) holds that high nitrogen affects the energy distribution of primary or secondary metabolism in plants. High-nitrogen environments induce plants to invest more resources in growth and inhibit the synthesis of secondary metabolites (Zheng et al., 2021). The expression of the plant defense response induced by herbivores may also be affected by N availability (Ren et al., 2013).

Ammonium and nitrate are the main forms of inorganic nitrogen that plants absorb. Nitrogen availability can change the basic biochemistry and other physiological activities of plants by regulating the primary metabolism of nitrogen and carbon (Wang et al., 2019). Ammonium is usually transformed into amino acids in plants, a process is called nitrogen assimilation(Ghanem et al., 2011). Amino acids, which are the first stable products of nitrogen assimilation, are significantly affected by the nitrogen regime and nitrogen source (Kant et al., 2011). As an important product of nitrogen assimilation, glutamic acid is a very active amino acid, being the starting point for the synthesis of other amino acids and can be rapidly converted into other nitrogenous compounds in plant cells. Glutamic acid metabolism plays a central role in plant nitrogen metabolism, including the biosynthesis of amino acids with key roles in plant defense, such as γ-aminobutyric acid (GABA) (Kan et al., 2015). Gamma-aminobutyric acid, a four-carbon nonprotein amino acid, exists in many organisms. In plants, GABA is considered a new plant growth regulator (Li et al., 2023). GABA can promote growth and improve plant resistance to stress by participating in signal transmission, regulating the stability of the plant antioxidant system, promoting photosynthesis and maintaining carbon and nitrogen metabolism(Shelp et al., 1999, Li and Zhou, 2022).

The entanglement between primary metabolism regulation and defense responses is a puzzling theme in plant science. Although many studies have focused on the effects of nitrogen on plant metabolism and the biological indicators of insect populations, the mechanism of metabolic changes in plant-insect interactions mediated by high nitrogen remains unclear. We hypothesized that high nitrogen can promote insect herbivore by changing the primary metabolism of plants. Tomato is an important crop and an excellent model plant for plant physiology research (Thaler et al., 2012). The oriental fruit fly, Bactrocera dorsalis (Hendel) (Diptera: Tephritidae), is a famous invasive agricultural pest that has caused serious losses in the global agricultural economy (Vargas et al., 2015). The main goal of this study is to address how high nitrogen affects the interaction between tomato and B. dorsalis by using metabolomics technology.

Some studies have reported that a high nitrogen supply increases the free amino acids in plants(Zhou et al., 2022). Plants use GABA, asparagine and arginine as nitrogen storage and transport amino acids (Kan et al., 2015). In particular, asparagine and arginine have higher nitrogen-to-carbon ratios and release and remobilize nitrogen after being hydrolyzed when the plant needs to mobilize nitrogen from source tissues (Hildebrandt et al., 2015). This study found that excess nitrogen promoted the biomass accumulation of tomato and B. dorsalis. The reason why high nitrogen increased the aboveground biomass of tomato was explained by the metabolomic results. First, it promoted primary metabolism (Atilio and Causin, 1996) and the accumulation of amino acids to promote the production of nutrients. We also found that key metabolites were significantly upregulated in the nitrogen excess treatment, particularly GABA and IAA. GABA connects the carbon and nitrogen metabolic fluxes in plants through the GABA shunt (Fait et al., 2008). Several studies have shown that GABA can promote pollen tube elongation and stem growth at low concentrations (Bouché and Fromm, 2004). In addition, IAA increases the accumulation of plant biomass. Ammonia nutrition can induce the activity of aldehyde oxidase and promote the synthesis of IAA (Koshiba et al., 1996).

As an inhibitory neuromuscular transmitter, GABA affects the normal development of insects by directly acting on GABA-gated chloride channels (Bown et al., 2006). The negative effects of GABA on insects have been verified in several studies. One study reared larvae with artificial diets containing GABA, and the feeding results showed that GABA significantly inhibited the growth of olive-banded leafler (Choristoneura rosaceana) larvae (Ramputh and Bown, 1996), and in another it significantly reduced the larval weight of Spodoptera littoralis (Scholz et al., 2015). The results of our research also showed that GABA had an inhibitory effect on the pupal weight of the fruit fly, and with increasing GABA concentration, the inhibition of biomass was more significant. Given the result of the upregulation of GABA content in nitrogen-excess fruits, ingestion of plant material that contains elevated GABA levels, could have negative consequences for insects (Bown et al., 2006). The growth of fruit fly larvae growing in nitrogen-excess fruits would in that case be inhibited, but the results were the opposite. Therefore, we also measured the metabolic changes of fruits that were cultured under different nitrogen levels with B. dorsalis injury to try to explain the reason for the change in biomass.

Experimental evidence supports the synthesis of GABA induced by herbivorous harm and involvement in plant defense (Bown et al., 2002; Mithofer and Boland, 2012). In plants, GABA biosynthesis is catalyzed by glutamate decarboxylase (GAD) (Akihiro et al., 2008). Plants initiate the signal transduction pathway when under environmental and biological stress, in which increased cytosolic Ca²⁺ stimulates Ca²⁺/calmodulin-dependent GAD activity and GABA synthesis (Zik et al., 2006). Research has demonstrated that GABA accumulation is stimulated by Spodoptera littoralis herbivory (Scholz et al., 2015). The crawling mechanical injury of Choristoneura rosaceana and Heliothis virescens can also induce the accumulation of GABA in a short time (Bown et al., 2002). GABA and glutamine are both downstream metabolites of glutamate metabolism. Glutamine synthetase (GS) catalyzes the synthesis of glutamine from free ammonium and glutamate. Some studies have shown that high-nitrogen nutrition can promote the activity of GS (Balotf and Kavoosi, 2016), and the assimilation of ammonium into glutamine is also one of the metabolic pathways which plants use to avoid the toxicity of high ammonium (Masclaux-Daubresse et al., 2006). Nitrogen-excess treatment provides tomato plants with a high concentration of ammonium, which is potentially toxic to plant cells. Free toxic ammonium in plants should be assimilated quickly to avoid ammonium toxicity (Linka and Weber, 2005). The main mechanism of ammonium assimilation is the GS/glutamine oxoglutarate aminotransferase (GOGAT) cycle. Ammonium can be assimilated into glutamine and glutamate via this cycle, which is the major pathway for primary nitrogen assimilation in plants (Lea and Miflin, 1974).

The metabolism of glutamate was the same in different treatment groups. The major reason is that plants seem to balance nitrogen metabolism by maintaining the homeostasis of glutamate. The concentration of glutamate in plants always remains relatively constant under different nitrogen levels in different culture conditions or in the diurnal cycle (Forde and Lea, 2007). High nitrogen input causes fluctuations in amino acids, including GABA, glutamine, arginine, asparagine, etc., in addition to glutamate. These metabolic changes are related to enzymes of nitrogen metabolism. Masclaux-Daubresse et al. (2006) argued that GS and glutamate dehydrogenase (GDH) were the major enzymes responsible for maintaining a constant concentration of glutamate (Masclaux-Daubresse et al., 2006), but it is also likely that the supply of 2-oxoglutarate is a key regulator (Forde and Lea, 2007). These studies explained that under high-nitrogen conditions, the content of glutamate in tomato fruit did not fluctuate, but the metabolism of GABA and glutamine fluctuated differently.

Glutamine is an important amino acid with the function of storing nitrogen, and an ammonium ion is fixed in the metabolic process of producing glutamine. B. dorsalis herbivory increases the levels of glutamate acid, phenylalanine and aspartic acid in tomato fruits with suitable nitrogen (Li et al., 2023). Our metabolomic results showed that injury to B. dorsalis changed the metabolic direction of glutamate and induced the upregulation of glutamine in the NE treatment group. The change in metabolic direction weakens the defense of tomato against B. dorsalis feeding. These results explain why the pupal weight of the fruit fly was increased in nitrogen-excess fruit that was injured by B. dorsalis. We believe that under the high nitrogen level, the change in glutamate metabolic direction was mediated by B. dorsalis, which may be the result of the joint action of carbon metabolism and nitrogen metabolism. Some reports have indicated that ammonium assimilation is closely related to carbon metabolism (Gauthier et al., 2010). The injury by B. dorsalis affected the carbon metabolism of plants, and high nitrogen affected their nitrogen metabolism, which destroyed the balance between carbon and nitrogen metabolism. Carbon metabolism provides the necessary energy and carbon framework for nitrogen metabolism. α-Ketoglutarate, the product of glucose metabolism and the TCA cycle, is an important carbon material in ammonium assimilation (Xun et al., 2020). GDH and GOGAT both catalyze the synthesis of glutamate from α-ketoglutarate, introducing a carbon framework into nitrogen metabolism (Masclaux-Daubresse et al., 2006). Some studies have shown that a sufficient carbon source allows plants to complete normal nitrogen metabolism and assimilate excessive ammonium (Vega-Mas et al., 2017). The damage by B. dorsalis significantly reduced the carbon source required for nitrogen metabolism. When the supply of the carbon framework is insufficient, plants need to seek efficient nitrogen assimilation pathways. Glutamine has a high nitrogen-to-carbon ratio (Kan et al., 2015); under this condition, ammonium assimilation is completed by glutamine synthetase. However, in the nitrogen-excess group, the KEGG pathway analysis did not show enrichment of the carbon metabolism pathway, possibly because the feedback of high nitrogen affects carbon metabolism. The results under nitrogen deficiency showed that the major differential metabolites were concentrated in carbon metabolism, and GABA was not a differential metabolite. Low nitrogen levels will significantly affect the carbon metabolism and nitrogen metabolism of plants, including the reduction of nitrogen-rich amino acid content (Zhou et al., 2021). Therefore, B. dorsalis did not induce significant metabolic changes in nitrogen-rich amino acids after harming nitrogen-deficient fruit. These results show that the GABA metabolome of plants defending against herbivorous harm depends on the level of nitrogen, making it a nitrogen-dependent defense response.

In conclusion, our research results supplement the research on the effect of high nitrogen on plant defense. Nitrogen excess promoted the aboveground biomass accumulation of tomato and B. dorsalis. According to our metabolomic results, fruits that are cultured in suitable nitrogen levels can promote the synthesis of GABA under biological stress. We also demonstrated the control effect of GABA on B. dorsalis. GABA supplementation not only increased the aboveground biomass of plants but also improved the defensive response of tomato. Injury by B. dorsalis inhibited the biosynthesis of GABA, but injury promoted the biosynthesis of glutamine in nitrogen-excess fruits ( Figure 8 ). The study showed that excess nitrogen inhibited the defense response of tomato to biological stress. These findings provide a foundation for future research on the nitrogen-mediated interaction between insects and plants.

The original contributions presented in the study are included in the article/ Supplementary Material. Further inquiries can be directed to the corresponding author.

Ethical review and approval was not required for the study on animals in accordance with the local legislation and institutional requirements.

HaL, YZ, and ZZ contributed to conception and design of the study. HaL organized the database. HaL and YZ performed the statistical analysis. HaL wrote the first draft of the manuscript. YZ wrote sections of the manuscript. All authors contributed to the article and approved the submitted version.